Continued Development of Techniques for In Vivo Investigation of the Function of Identified Neurons One of our major aims is to understand how circuitry within the basal ganglia contributes to the control and learning of actions. An important component of this line of research is gaining an understanding of the activity of specific neuronal subtypes within this circuitry during the learning, planning, initiation and performance of actions and action sequences. We have worked with Drs. Rui Costa of the Champalimaud Neuroscience Institute and Steven Vogel of the Laboratory of Molecular Physiology at NIAAA to develop a technique for in vivo fiber photometry to perform Time Correlated Single Photon Counting (TCSPC) fluorimetry in the striatum in vivo. In previous studies we used this technique to determine the activity of specific striatal neuronal subtypes during performance of an instrumental task. We are now extending this approach to measure calcium transients in a variety of neuronal somata and presynaptic elements within the cortico-basal ganglia circuitry. We have just completed a study in which we measured presynaptic calcium signals during skill learning on the rotarod task in both medial prefrontal cortex afferents to the dorsomedial striatum, and afferents from the primary motor cortex to dorsolateral striatum. We also used a viral approach to express the genetically-encoded calcium sensor GCaMP6 in the somata of neurons that give rise to these projections. By comparing the somatic and presynaptic alterations in calcium signaling during the learning and performance of skill, we have been able to determine the cellular locus of learning-related plasticity. We have also modeled the calcium responses to afferent stimulation using simultaneous in vivo fiber photometry and electrophysiological recording in anesthetized mice. We have also examined calcium transients in dopaminergic somata and afferents, as well as somata in other cortical regions. In addition, we are developing constructs to express other genetically-encoded molecular sensors in specific neuronal subtypes in different brain regions. Our goal is to measure how intracellular signaling changes as animals learn, and also to examine effects of acute and chronic ethanol exposure on this signaling. Alcohol and Cannabinoid Effects on Sleep Dysregulation of sleep is a common effect of a variety drugs of abuse, including alcohol. However, little is known about what aspects of sleep-related brain physiology are altered by phytocannabinoids (e.g. delta9-tetrahydrocannabinol, THC) and synthetic cannabinoids such as those present in spice compounds. Furthermore, the role of endocannabinoids in sleep regulation is not fully understood. To address these questions we implemented a two-region EEG recording system combined with EMG recording in mice to detect sleep-related physiology. We then developed a fully-automated sleep state assignment system with 2 sec resolution. Using these tools, we found that increasing endocannabinoid levels led to a short-lasting increase in non-REM sleep, characterized by increased length of non-REM sleep bouts. Similar effects were observed with a synthetic CB1 agonist, and a later-onset reduction of non-REM sleep time and stability was also observed. This latter reduction in non-REM sleep was mimicked by blockade of CB1 cannabinoid receptors, which essentially fragmented sleep. This finding indicates that endocannabinoids normally act to stabilize non-REM sleep through CB1 receptor activation. We are currently examining which sleep-related brain regions contribute to these cannabinoid/endocannabinoid actions. Effect of chronic THC exposure on sleep and sleep architecture are also being examined. We have also examined effects of acute and chronic ethanol exposure on sleep and sleep architecture. Insomnia is a prominent effect of abstinence/withdrawal in humans with alcohol use disorders, and ethanol may be used in an effort to restore or improve sleep. We examined measured sleep periods up to 72 hours in mice using the techniques described above. A single acute exposure to ethanol had minimal effects on sleep architecture at doses up to 2 g/kg. However, administration of a 4 g/kg dose induced a state that had features of both the waking state (i.e. clear bouts of EMG activity) and non-REM sleep (i.e. low frequency EEG activity), in addition to elevated theta frequency EEG activity that is not characteristic of either state, which persisted for 2 hours after ethanol dosing. This unique, high alcohol dose-induced state also appears to differ from EEG patterns observed with most general anesthetics. We are currently interested in determining what neurotransmitter systems contribute to this odd signature of strong intoxication. We also exposed mice to chronic ethanol using the vapor inhalation model. We found reduced non-REM sleep and fragmented non-REM sleep bouts at the end of this chronic exposure. During acute withdrawal after this exposure, mice continued to show reduced non-REM sleep and the duration of non-REM sleep bouts was reduced. We are now examining the duration of these changes following the offset of the chronic exposure paradigm. We also hope to examine which neurotransmitters and brain regions have important roles in the non-REM sleep disruption by ethanol. Ultimately, it will be helpful to determine if restoring normal non-REM sleep will reduce the enhanced ethanol intake observed following this chronic ethanol exposure paradigm. We have also developed an automated and programmable system for sleep disruption that we can use to determine how drugs interact with explicit sleep disruption or insomnia.